Concerted evolution of metabolic rate, economics of mating, ecology, and pace of life across seed beetles

Significance Coevolution between females and males has led to remarkable differences between the sexes but has taken very different routes, even in closely related animal species, for reasons that are not well understood. We studied the physiological processes that convert resources into offspring (metabolism) in males and females of several related beetle species. We found that ecological factors dictate metabolic rate, which, in turn, have predictable direct and indirect effects on male–female coevolution. Our findings suggest that a complete understanding of differences between the sexes requires an understanding of how ecology affects metabolic processes and how these differ in the sexes.

discarded as a burn-in. Thus, each observation consisted of four repeated measures of the amount of CO 2 produced and O 2 consumed during 60 min by an individual beetle and the cumulated amount of activity performed during this time.
All individuals were ca 24 hrs old virgins at the start of the experiment. Half of all individuals were allowed to mate immediately before being placed in a respirometry chamber while the other half remained virgin. All individuals were weighed to the nearest 0.01 mg using an electronic microbalance (Sartorius Genius ME 235P-OCE) prior to the experiment, and all males in the mated treatment were weighed both before and after mating to determine ejaculate weight (as the difference in body weight). Our experimental design thus had 48 cells (12 species × 2 sexes × 2 mating status) and the average sample size per cell was 15.7 individuals (total N = 753).
Measures of metabolic rate are often confounded by an unknown degree of variation in activity during recording (1). Here, we used activity-compensated measures of CO 2 and O 2 for all downstream analyses. For each cycle, species and sex, we regressed both CO 2 and O 2 on activity and added the residual to the respective intercept. This yields the value expected under zero activity for all observations (i.e., resting metabolic rate), and will equal the observed value for all individuals showing zero activity. The overall correlation between uncompensated and compensated CO 2 and O 2 values was r = 0.93, suggesting that on average 14% of total variance was due to variation in activity across individuals. We assessed sex and species specific scaling in metabolic rate by fitting the function CO 2 = c + body mass m , by means of non-linear regression using data on virgin individuals. The average exponential scaling coefficient was m = 1.12 in females and m = 1.17 in males, but in no species did the exponential scaling coefficient differ between the sexes within species (P > 0.054 in all cases). In only four out of 24 cases did the 95% CI of m not overlap with m = 1 within species. Here, we thus follow convention (1) and use activity compensated values of CO 2 and O 2 per mg body weight (i.e., mass-specific RMR) as our measure of RMR and the ratio CO 2 produced /O 2 consumed as our measure of RQ in all comparative analyses.
An analysis of the full data set showed a strong general effect of mating status on metabolism, which differed in magnitude between species and between sexes. Here, we thus used the marginal mean per cell of our design, averaged over all four cycles, to characterize the species, sex and treatment specific metabolic metrics. For each sex, we derived four species specific measures of weight specific metabolic parameters. (1) RMR and (2) RQ in virgin individuals, to provide baseline metabolic parameters. (3) RMR mated -RMR virgins and (4) RQ mated -RQ virgins were used to characterize mating-induced changes in metabolism.
Cost of mating in males. Virgin males and females were collected upon emergence from beans and isolated individually. Males were randomly assigned to one of two treatments, one in which males were precluded from reproductive interactions and one in which males mated and competed against other males freely. In treatment A, each male was placed alone in a petri dish for life. In treatment B, three males and three females were introduced together for life in a petri dish. In both treatments, males were weighed prior to the experiment and all dishes were provided with an ample supply of beans. We performed spot checks at least once per 24 hrs to determine male life span. We conducted 16 -20 replicates per treatment level and species (total N = 457 males).
The data from this experiment was used to estimate two metrics. Cost of mating and reproduction in females. Females of some species of seed beetles suffer costs of male harassment (2,3) so in order to isolate the economic effects of mating in females we did not employ male-female cohabitation but staged all matings. Virgin males and females were isolated individually upon emergence and all individuals used were young and had just entered adult reproductive maturation when assigned to a treatment (i.e., 1-3 days old in all species but in M. dorsalis, M. tonkineus and A. robiniae where individuals were 5-7 days old). Females generally do not lay eggs in the absence of host beans and to separate the economic effects of mating itself from the cost of reproduction, females were thus assigned to either of the following four treatments; (A) virgin females were isolated in an empty 9 cm Ø petri dish (i.e. no mating or reproduction), (B) females were mated once and then isolated in an empty petri dish (i.e. mating once but no reproduction), (C) females were mated once and then isolated in a petri dish containing beans (i.e. mating once and reproduction), (D) females were mated twice (day 1 and 3) and were from the first mating isolated in a petri dish containing beans (i.e. mating multiply and reproduction). Matings were staged by introducing a virgin male to the female. All matings were monitored and males removed from vials following copulation. Females in treatment C and D were provided with a superabundant supply of host beans (21 g). Females were weighed to the nearest 0.01 mg using an electronic microbalance (Sartorius Genius ME 235P-OCE) prior to the experiment, and all males weighed both before and after mating to determine ejaculate weight (as the difference in body weight).
We conducted on average N = 17.6 replicates per treatment level and species (range 8 − 27; total N = 843 females). Female life span (spot checks ≥1 per day) and reproductive effort (the number of eggs dumped in petri dish in treatment A and B, number of offspring produced in treatment C and D) were recorded in all replicates. In treatment groups C and D, a few females that laid fewer than 5 eggs in their lifetime were omitted from further analysis, as they had likely not copulated successfully (N = 54 in total; N = 45 of these laid zero or only a single egg) and inclusion of these would bias our downstream analyses. We then estimated species and treatment level specific average life span and average fecundity for level C and D from this experiment.
For our estimates of the cost of mating and the cost of egg production, the treatment levels A and B should represent female life span in the absence of the costs of egg production.
However, some females in A laid a few eggs despite them being virgin (i.e., egg dumping) and some females in B laid a few eggs despite the absence of larval resources (i.e., beans).
Because female life span is negatively related to egg production (SI Appendix, Fig. S2), the raw life span means are less than ideal. For these two levels, we therefore estimated female life span compensated for egg production for females in treatment levels A and B. For each species, we performed a linear regression of life span on the number of eggs laid, and for each female we then summed the residual with the species specific intercept. For females laying no eggs, this equals their observed life span. For those females that laid a few eggs, this yields the predicted life span that each female should have had if she had laid no eggs.
We then estimated species and treatment level (for A and B) specific average life span using these compensated values. We note that they differed only very marginally from the raw uncompensated estimates (difference on average 3.8%).
The data from this experiment was used to estimate six species-specific metrics. Copulation duration, defined as the time from full male genital insertion to male-female separation, was recorded for all matings. All females were subsequently dissected under a Leica M165 C stereo microscope and several photos were taken of internal reproductive anatomical traits using a Lumenera Infinity 2-5C digital microscope camera, under standardized setting and lighting.
Images were analysed using Infinity Analyze 6.1. The bursa copulatrix (BC) is semitransparent and the area of the ejaculate was recorded as seen through the dorsal wall of the intact BC (SI Appendix, Fig. S3). The area of the BC itself was recorded following dorsal slitting and spreading of the BC, placed under a microscope slide. We also recorded the area of the spermatheca, the presence of chitinised oval rings and a tooth on the wall of the BC (SI Appendix, Fig. S3) and, finally, the length of the elytra to provide a measure of general female body size.
To characterize the ejaculate processing rate of each species (SI Appendix, Fig. S4), we first calculated ejaculate volume based on ejaculate area, using a spherical approximation. resulting from 15 source trees based on mitochondrial and nuclear genetic data as well as morphology. The topology of this phylogenetic hypothesis is very well supported indeed (5,6). To assess phylogenetic signal in our data, we estimated both Pagel's λ (7) and Blomberg's K (8) for all traits. For both metrics, values of zero indicates that traits have evolved independently of the phylogeny (i.e., close relatives are not more similar than more distant relatives) while unity represents a strong phylogenetic signal consistent with trait evolution according to a Brownian motion model. We tested for correlated evolution both through the use of PICs (9) and through phylogenetic generalized least squares (PGLS) models of trait evolution (10), using the ML estimate of λ. Phylogenetic comparative analyses were performed using ape v.5.5 (11) and phytools v.0.7-90 (12), in R v. 4